Hydroxylamine compositions for the prevention or retardation of cataracts

ABSTRACT

A pharmaceutical composition and treatment to inhibit the development of cataracts in the crystalline lens of the eye by administering a hydroxylamine to a subject at risk of developing a cataract. The pharmaceutical composition comprises a hydroxylamine compound in a therapeutically sufficient amount to prevent or retard the development of the cataract. A reducing agent can also be administered in combination with the hydroxylamine. Particular examples of the hydroxylamine are TEMPOL-H, TEMPO-H and OXANO-H, while particular examples of the reducing agent are a sulfhydryl compound, such as N-(2-mercaptopropionyl)glycine (MPG), N-acetyl cysteine, β-mercaptopropionyl glycine, and glutathione. In particular embodiments, the composition comprises TEMPOL-H in an amount that is sufficient to provide a concentration of about 1 μM to 1 mM in the aqueous humor of the eye, and mercaptopropionyl glycine in an amount sufficient to provide an aqueous humor concentrations of about 0.1 to 5 mM.

This case claims benefit of Provisional Application 60/010,637 filedJan. 26, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compositions for the prevention andtreatment of cataract formation in the crystalline lens of the eye

2. Discussion of the Background

Aging-related cataract is a gradual opacification of the crystallinelens of the eye, which is presently treated by surgical removal andreplacement of the cataractous lens. Cataracts are believed to be adisease of multifactorial origin involving many of the same processesthat characterize the process of aging in other tissues. Dataaccumulated over a period of years from the work of many laboratoriesindicates that once begun, cataract development probably proceeds viaone or more common pathways or processes that culminate in damage tolens fibers. Since cataract is already a slowly progressing diseasewhich occurs predominantly in the elderly, a significant retardation ofits rate of development could eliminate the need for many surgicalcataract extractions. This reduction would provide tremendous benefitsboth to individual patients and to the public health system.

Based on research from the inventors and others, several processes havebeen proposed as crucial factors in cataractogenesis. These processesinclude oxidation, phase separation phenomena and proteolysis. With thehypothesis that one or more of these processes probably represent"common pathways" involved in lens opacification, the inventorsinitiated an effort to screen compounds which might inhibit theseparticular processes for their potential as anti-cataract agents. Inkeeping with the likelihood that the disease is multifactorial, theinventors tested the agents not only individually but in variouscombinations as well.

One active area of medical research has been an investigation of therole that free radical scavengers and antioxidants may play in theprevention and treatment of diseases caused by oxidative stress. Thefocus of many such investigations has been upon biochemical pathwaysthat generate reducing power in cells, for example, glutathionesynthesis and cycling. Enzymes that reduce activated oxygen species,such as superoxide dismutase, have also been studied to determinewhether they diminish cellular oxidative stress. Compounds forinhibiting lipid oxidation in cell membranes by direct radicalscavenging have also been considered to be promising therapeuticinterventions. The administration of compounds such as vitamin E,carotenoids, selenium compounds and vitamin C (ascorbate), for theirantioxidant effects, is known in the popular culture.

There are many reviews in the literature of in vitro and clinicalstudies of the medical effect of antioxidants and free radicalscavengers (references 1-3, below). Thiol compounds have been ofparticular scientific interest, because glutathione cycling plays a rolein maintaining the redox balance in cells. Selenide compounds havingglutathione peroxidase activity, for example, are the subject of U.S.Pat. No. 5,321,138. Thiol derivatives of amino acids have also beenstudied as antioxidants. In particular, mercaptopropionyl glycine (MPG)has been investigated for it effect in reducing cataract formation (4).

In the present invention, the nitroxide family of radicals is ofparticular interest. Nitroxides are free radicals that are stable, andwhich are reducible to their corresponding hydroxylamines. Nitroxideswere originally of interest to physical chemists due to theirparamagnetic properties, allowing their use as "spin-labels" in electronparamagnetic resonance studies. These compounds have more recently beenstudied because of their radical scavenging properties; nitroxides mimicthe enzymatic activity of superoxide dismutase (5-8). Nilsson et al.disclosed, in WO 88/05044, that nitroxides and their correspondinghydroxylamines are useful in prophylaxis and treatment of ischemic celldamage.

Reddan et al. (9) have investigated the use of the nitroxide TEMPOL toprotect lens epithelial cells from hydrogen peroxide damage in vitro.Mitchell et al., in U.S. Pat. No. 5,462,946, also disclose use ofnitroxides (such as TEMPOL) to protect lens epithelial cells fromoxidative damage. However Mitchell and his colleagues have also reportedthat only the nitroxide TEMPOL protects cells from oxidative damage, andthat the corresponding hydroxylamine TEMPOL-H (the reduced nitroxide)affords no such protection (12, 13).

In spite of years of sustained study into the cause and treatment ofcataracts, a clinically useful non-surgical treatment that retards thedevelopment of age-related cataracts has eluded researchers.Sorbitol-lowering drugs (aldose reductase inhibitors) have been found tohave some effect in retarding the development of cataracts in rats withhigh galactose intake. Aspirin, acetaminophen and ibuprofen have alsobeen shown to delay experimental cataracts. Bendazac has been found toprotect lens proteins in vitro, and to delay the onset of cataracts inx-ray irradiated rats. Yet other treatments that have been proposed forthe treatment of cataracts include vitamins, aminoguanidine, and variousherbal preparations. None of these treatments has yet been demonstratedto be clinically useful.

It is therefore an object of this invention to provide a clinicallyuseful, non-surgical treatment to retard or prevent the development ofcataracts in the crystalline lens of the eye.

This and other objects of the invention will be understood more clearlyby reference to the following detailed description and drawings.

SUMMARY OF THE INVENTION

The present invention resides in the surprising finding that ahydroxylamine is a better anti-cataractogenic composition than thecorresponding nitroxides. This finding is contrary to the teaching ofthe prior art that hydroxylamines provide much less protection againstoxidative damage than corresponding nitroxides. The pharmaceuticalcompositions of the present invention include a hydroxylamine, or ahydroxylamine and a reducing agent that opposes formation of nitroxidesfrom the hydroxylamines, for prevention and/or treatment of cataracts.

The pharmaceutical composition includes a hydroxylamine compound in atherapeutically sufficient amount to prevent or retard the developmentof a cataract in a subject to whom the composition is administered. Thecomposition may further include a reducing agent for maintaining thehydroxylamine substantially completely in a reduced form. The reducingagent may be a sulfhydryl compound that is present in a sufficientamount to maintain substantially all the hydroxylamine compound in areduced state.

In specific embodiments, the reducing agent is selected from the groupconsisting of mercaptopropionyl glycine, N-acetyl cysteine (NAC),β-mercaptoethylamine and glutathione, and the hydroxylamine compound isselected from the group consisting of TEMPOL-H (the hydroxylaminereduced form of the nitroxide4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy), TEMPO-H (thehydroxylamine reduced form of the nitroxide2,2,6,6-tetramethylpiperidin-1-yloxy) and OXANO-H(2-ethyl-2,4,4-trimethyloxazolidine, which is the reduced form ofOXANO⁻, 2-ethyl-2,4,4-trimethyloxazolidin-3-yloxy). In the particularpreferred embodiment, the sulfhydryl compound is N-(2-mercaptopropionyl)glycine (hereinafter mercaptopropionyl glycine or MPG) and thehydroxylamine compound is TEMPOL-H.

The composition may be a topical preparation or dosage unit formsuitable for instillation in the human eye. Such a composition mayinclude TEMPOL-H in an amount that is sufficient to provide TEMPOL-H ina blood concentration of about 25 μM to 3 mM, and mercaptopropionylglycine in an amount sufficient to provide mercaptopropionyl glycine ina blood concentration of about 0.25 to 50 mM. Alternatively, thecomposition includes TEMPOL-H in an amount that is sufficient to provideTEMPOL-H in a concentration of about 1 μM to 1 mM in the aqueous humorof the eye, and mercaptopropionyl glycine in an amount sufficient toprovide mercaptopropionyl glycine in a concentration of about 0.1 to 5mM in the aqueous humor of the eye.

The invention also includes a method for inhibiting or preventing thedevelopment of a cataract, or inhibiting the progression of an incipientcataract, which comprises administering to a patient a hydroxylaminecompound and a reducing agent in a therapeutically sufficient amount toprevent or retard the development of a cataract in a subject to whom thecomposition is administered. In specific embodiments of the method, thehydroxylamine compound is TEMPOL-H and the reducing agent ismercaptopropionyl glycine. The hydroxylamine and reducing agent may beadministered topically, systemically, or intraocularly, and eitherseparately or in combination. Prodrug forms of either the hydroxylamineor reducing agent may be administered, for example to increase cornealpenetration. The prodrug could for example be a nitroxide, which issubsequently converted to a hydroxylamine by the reducing agent.

Topical dosage forms include liquid eye drop preparations that may beinstilled externally to the eye, or adsorbed into a material such as asoft contact lens or a collagen corneal shield. The hydroxylamine andreducing agent may be introduced into the eye, either separately or incombination, using such forms as topical drops, ointment, periocular(for example subconjunctival) injection, or intraocular instillation(for example by implantation of an intraocular reservoir). Thehydroxylamine (and reducing agent if desired) may also be administeredorally, in a sufficient amount to raise levels of the drug to desiredamounts in the blood or aqueous humor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effect of H₂ O₂ treatment on rubidiumaccumulation by cultured rat lenses.

FIG. 2 is a bar graph showing amelioration of H₂ O₂ inhibition oftritiated choline accumulation by cultured rat lenses as affected bycarnosine, MPG and TEMPOL-H.

FIG. 3 is a graph showing the lack of interaction of H₂ O₂ with TEMPOL-Hin culture medium.

FIG. 4 is a structural drawing showing the redox cycle between TEMPOLand TEMPOL-H.

FIG. 5 is a bar graph showing a dose-response effect of TEMPOL-H, andthe combination of TEMPOL-H and MPG, in ameliorating H₂ O₂ inhibition ofcholine accumulation by cultured rat lenses.

FIG. 6 is a bar graph showing the dose-response effect of TEMPOL-H, andthe combination of TEMPOL-H and MPG, in ameliorating H₂ O₂ inhibition ofrubidium accumulation by cultured rat lenses.

FIG. 7 is a photograph showing opacification of cultured rat lenses byH₂ O₂ treatment and inhibition of opacification by TEMPOL-H.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical compositions of the present invention include ahydroxylamine compound, or the combination of the hydroxylamine compoundand a reducing agent. The hydroxylamine component can be anyphysiologically acceptable hydroxylamine. Preferable hydroxylaminecompounds are monocyclic hydroxylamines wherein the monocycle is anoxazolidine, pyrrolidine or piperidine. Especially preferredhydroxylamines are TEMPO-H , TEMPOL-H and OXANO-H. These compoundspossess either a 2,2,6,6-tetramethylpiperidin-1-yloxy structure (thepiperidine nitroxide radicals TEMPO⁻ and TEMPOL⁻ which are reduced tothe corresponding hydroxylamines TEMPO-H and TEMPOL-H), or the4,4-dimethyloxazolidin-3-yloxy structure (the oxazolidine nitroxideradical OXANO⁻ that is reduced to the corresponding hydroxylamineOXANO-H).

The reducing agent component of the compositions of the inventionfunctions to maintain the reduced state of the hydroxylamine and/or toprovide reducing equivalents directly to the cells of the lens. Thereducing agent can be a small organic molecule. Preferable small organicreducing agents are the class of sulfhydryl compounds, such asglutathione. Preferred sulfhydryl compounds are substituted mercaptoamino acids, such as mercaptopropionyl glycine. Other sulfhydrylcompounds including N-acetyl cysteine, β-mercaptoethylamine andglutathione can also be combined with the hydroxylamine. Precursors ofN-acetyl-cysteine can also be used, for exampleS-isobutyrl-N-acetyl-L-cysteine ethyl ester and other compoundsdisclosed in U.S. Pat. No. 5,519,054 as prodrugs of N-acetyl-cysteinethat are quickly delivered across the cornea

Additional compounds of interest can be tested for theircataract-inhibitory activity by testing the composition in the lensorgan culture system described in detail in references 10 and 11, andExample 1.

The compositions of the invention are useful for preventing cataractformation, or for treating cataracts, especially by slowing theirprogression. The compositions can be formulated forprophylactic/therapeutic administration by methods typical in the art,maintaining required sterility and osmolarity. Preferred methods ofadministration are as eye drops or as an addition to an ophthalmicointment or lubricant (e.g. DURATEARS, Alcon Ophthalmic). Thecompositions of the invention can also be adsorbed into dehydrated softcontact lenses (e.g. Lidofilcon B, Bausch & Lomb CW79 or DELTACON(Deltafilcon A)). Alternatively, supports such as a collagen cornealshield (e.g. BIO-COR dissolvable corneal shields, Summit Technology,Watertown, Mass.) could be employed. The compositions can also beadministered by infusion into the eyeball, either through a cannula froman osmotic pump (ALZET, Alza Corp., Palo Alto, Calif.) or preferably byimplantation of timed-release capsules (OCCUSENT) or biodegradable disks(OCULEX) which contain the compositions. These routes of administrationhave the advantage of providing a continuous supply of the compositionto the eye.

A preferred method of administration will be one which provides forcontinuous administration to the eye, preferably into the aqueous humor,so that the lens is directly treated. Such methods as delivery bycannula or osmotic pellets are presently most useful, because of theirunambiguous delivery to the anterior chamber, in initial testing of thecomposition. However, as it is expected that long-term, treatment isneeded to provide effective therapy, preparations which can beadministered topically, i.e. as eye drops or ointments, are mostpreferred.

The composition is formulated and administered so as to apply a dosageeffective for alleviating oxidative stress in the lens of the eye,and/or inhibiting the development of cataracts in the eye. Theconcentration of the hydroxylamine component will preferably be in therange of 1 μM to 1 mM in the aqueous humor. Most preferably the range ofhydroxylamine concentration will be from 10 to 100 μM. The concentrationof the reducing agent will be from 0.1 to 5 mM in the aqueous humor,preferably in the range of 0.5 to 2 mM. The concentrations of thecomponents of the composition are adjusted appropriately to the route ofadministration, by typical pharmacokinetic and dilution calculations, toachieve such local concentrations. If the composition is administeredsystemically, it is estimated that concentrations of 25 μM to 3 mM ofthe hydroxylamine component in the blood will be appropriate. Acorresponding unit dosage would be 10 to 1200 mg of hydroxylamine per100 kg body weight. Similarly, systemic administration should seek toachieve a blood concentration of the reducing agent component of 0.25 to50 mM, corresponding to a unit dose of 100 to 6,000 mg per 100 kg bodyweight.

Particular embodiments of the invention are illustrated by the Examplesset forth below.

EXAMPLE 1

Effect of H₂ O₂ on Accumulation of Rubidium-86 in Rat Lens

In order to quantitate the effect of drugs that affect cataractdevelopment, most assays have used membrane transport (i.e. the abilityof the lens to actively transport radiolabelled compounds from themedium) as the test parameter. Normal lenses actively accumulate aminoacids, choline or rubidium-86 (Rb-86) from the medium, and the lensesexhibit decrements in such accumulation when the lens epithelium isstressed. Membrane transport assays provide a very sensitive measurementof the health of the lens, and the results indicate changes in lensphysiology well before obvious opacification begins. Such assays areaccepted as an indication of the in vivo effect of a drug in retardingthe development of cataracts.

The techniques described in references 10 and 11 were used in thisassay. Rat lenses used in these studies were obtained fromSprague-Dawley male rats. The lenses were incubated in 24-well clustersin modified TC-199 medium (prepared as described in Zigler and Hess,Exp. Eye Res. 41:67, 1985) and placed in a 37° C. incubator with a 95%air/5% CO2 humidified atmosphere. The lenses were incubated in about 2.0ml of culture medium, which was adjusted to about 300 milliosmoles. Theassays were usually of 22 hours duration, and tracer levels of ⁸⁶ Rb ortritiated choline were added at 18 hours so that the tracer was presentduring the last four hours of the assay.

FIG. 1 gives the results of a representative experiment in which the ratlenses were stressed in organ culture by addition of a bolus of H₂ O₂ tothe medium at the beginning of the assay, equalling a finalconcentration of 0.25 mM in the initial cultured medium. The normalcontrol lenses were able to accumulate Rb-86 to an internalconcentration more than 7-fold greater than the concentration in themedium while those lenses exposed to H₂ O₂ could accumulate it to onlythe 5-fold level. Two potential anti-cataract agents were tested in thisexperiment: E-64 (a protease inhibitor) and pantetheine (a putativephase separation inhibitor) were added to the culture medium at thebeginning of the assay. Both appeared to have a small negative effect onRb-86 accumulation when added to the medium of control lenses andneither prevented the decrement in accumulation associated with H₂ O₂exposure.

EXAMPLE 2

TEMPOL-H and MPG Inhibit Cataractogenesis by H₂ O₂

MPG is a sulfhydryl compound and reducing agent which may be acting inpart by directly removing H₂ O₂ from the medium. TEMPOL-H does not reactwith H₂ O₂, as shown by the assay of the concentration of H₂ O₂ inmedium containing H₂ O₂ with and without TEMPOL-H (see the open circleline in FIG. 3). This data indicates that TEMPOL-H is effectiveintraocularly, i.e. it protects the lens after being taken up into thecells of the lens, particularly the lens epithelium.

TEMPOL-H is the reduced form of the nitroxide TEMPOL, which is a stablefree radical detectable by electron paramagnetic resonance (EPR)spectroscopy, as in reference 12. TEMPOL-H can be readily converted toTEMPOL by oxidation with ferricyanide, and then can be quantitated byEPR spectroscopy. This provides a ready means of determining whetherTEMPOL-H is present in experimental lenses and whether it is in theoxidized or reduced state. FIG. 4 shows the structure of both compoundsand some agents which can effect the redox conversions. That Figureshows that the nitroxide TEMPOL can be reduced to the correspondinghydroxyl TEMPOL-H by reduced glutathione (GSH), ascorbate andreductases. TEMPOL-H can in turn be oxidized to the nitroxide bydehydrogenases and activated oxygen species, such as singlet oxygen.

Rat lenses were cultured as described in reference 11 and Example 1. H₂O₂ was added to experimental cultures to a concentration of 0.25 mM.Carnosine at 1 mM, MPG at 1 mM, or TEMPOL-H at 5 mM was added to some ofthe treated lenses at the beginning of the culture assay. FIG. 2 showsthe results of the experiment. Both MPG and TEMPOL-H show significantreduction in the decrement of tritiated choline concentration caused bythe H₂ O₂ treatment.

Following lens incubation, lenses were rinsed, then homogenized inbuffer and aliquots of the homogenate analyzed by EPR. Results clearlydemonstrated that TEMPOL-H does penetrate the lens from the culturemedium and that the highly reducing environment within the lens keepsthe compound in the reduced state. Only following treatment of the lensextract with ferricyanide was the nitroxide detected.

EXAMPLE 3

Dose Response of TEMPOL-H and TEMPOL-H with MPG

FIGS. 5 and 6 provide further data on the effects of TEMPOL-H and MPG onaccumulation of ³ H-choline and Rb-86, respectively, in cultured lensesexposed to H₂ O₂. The experiment was performed essentially as describedfor Example 2, except that TEMPOL-H was added at 3 or 4 mM or was addedat 3 mM together with MPG at either 0.5 or 1 mM. The data demonstrate adose response effect for TEMPOL-H and indicate that the combination ofTEMPOL-H with MPG has greater efficacy than TEMPOL-H alone. However,even very low concentrations of TEMPOL-H, or TEMPOL-H and MPG, arebelieved to exert some protective effect against cataract formation.

EXAMPLE 4

TEMPOL-H Prevents Opacification of Cultured Lenses

Exposure of rat lenses to a bolus of 0.25 mM H₂ O₂, severely affectsmembrane transport parameters, but does not cause the lenses to becomeopaque. However, exposure to 1 mM H₂ O₂ does produce overt cataract.Lenses were cultured as in the above examples. FIG. 7 shows two controllenses (left pair) after 24 hours incubation in a medium that containsneither H₂ O₂ nor TEMPOL-H, two lenses incubated for 24 hours in mediumto which H₂ O₂ was added to a concentration of 1.0 mM at time zero(center pair), and two lenses incubated in 1.0 mM H₂ O₂ plus 4 mMTEMPOL-H (right pair). The TEMPOL-H clearly and consistently was foundto inhibit opacification of the cultured lenses, as shown in the rightpair of lenses in FIG. 7.

EXAMPLE 5

Method of Treatment

The present invention includes a treatment that inhibits the developmentof cataracts in a subject such as an animal, for example a rat, rabbit,dog or human. The method includes administering the hydroxylamine, or acombination of the hydroxylamine and a reducing agent, to the subject ina pharmaceutically compatible carrier and in an effective amount toinhibit the development of cataracts in the crystalline lens of the eye.Although the treatment can be used prophylactically in any patient in ademographic group at significant risk for cataracts (for example humansover the age of 55, or subjects of that age who smoke tobacco), subjectscan also be selected using more specific criteria. The treatment can beadministered, for example, to subjects who have biomicroscopic clinicalevidence of an incipient cataract (for example a nuclear scleroticcataract), or biomicroscopic evidence of a cataract combined with adecrease in visual acuity. Other types of cataracts that would betreated include steroid induced catracts (for example posteriorsubcapsular cataracts), diabetic cataracts, and cataracts induced byexposure to chemicals or radiation.

The administration of any exogenous TEMPOL-H (or other hydroxylamine)would inhibit the progression of a cataract as compared to a subject towhom the exogenous hydroxylamine was not administered. Theanti-cataractogenic effect, however, increases with the dose of thehydroxylamine. In some embodiments of the invention, sufficienthydroxylamine is administered to achieve a concentration in the aqueoushumor of at least 1 μM, more specifically at least 1 mM. In thisexample, this concentration can be achieved by direct intraocularinjection into the anterior chamber of the eye of a 100 mM sterilesolution of TEMPOL-H, or delivery of this amount by an implanted pumpthat delivers a daily intraocular dose of the drug, for example into theanterior chamber of the eye. Paracentesis of the anterior chamber canalso be initially or repeatedly performed to determine the concentrationof the hydroxylamine in the eye by subjecting the aqueous humor toelectron paramagnetic resonance (EPR) studies, as described in reference11. The information obtained by paracentesis and EPR would then be usedto modify the dosage given, or the frequency of administration.

EXAMPLE 6

Topical Delivery

A more clinically convenient treatment would be administration of thehydroxylamine in liquid eye drops, for example a 50 μL drop of a 100 mMsterile solution of the hydroxylamine TEMPOL-H. Alternatively, thehydroxylamine is administered by direct subconjunctival injection of 1-2ml of the sterile 100 mM solution, either daily or weekly. Injection ofthe medication beneath the conjunctiva or Tenon's capsule allows thedrug to substantially bypass the conjunctival and corneal epithelialbarriers. The eye drops can be formulated in a pharmaceutically inert,biologically acceptable carrier, such as isotonic saline or an ointment.Conventional preservatives, such as benzalkonium chloride, can also beadded to the formulation.

The active ingredient it typically dissolved in a buffered, isotonicsolution containing antimicrobial preservative agents. In ophthalmicointments, the active ingredient is admixed with a suitable base, suchas white petrolatum and mineral oil, along with antimicrobialpreservatives. Ophthalmic disks will typically be constructed to containa core of active ingredient surrounded by a polymer matrix, such as ahydrophobic ethylene/vinyl acetate copolymer. Specific methods ofcompounding these dosage forms, as well as appropriate pharmaceuticalcarriers, are known in the art. REMINGTON PHARMACEUTICAL SCIENCES, 16thEd., Mack Publishing Co. (1980).

EXAMPLE 7

Systemic Therapy

Experiments were performed with 150-250 gram Sprague Dawley albino ratsto demonstrate that systemic delivery of the hydroxylamine and reducingagent could provide intraocular doses of these drugs. A 2 ml volume of100 mM TEMPOL-H was provided in an ALZET osmotic pump, available fromAlza Corporation of Palo Alto, Calif. The pump was programmed to deliverthe 2 ml dose continuously (down an osmotic gradient) over a period ofeither 7 or 14 days. The tip of the pump's delivery cannula wasimplanted either subcutaneously (on top of the neck of the rat),intraperitoneally, or in the superior fornix of the eye between theupper eyelid and bulbar conjunctiva.

In the rats who received subcutaneous delivery of the hydroxylamine,plasma TEMPOL-H concentrations of 0.25 μM were observed. Concentrationsin the eye were found to be 0.02 μM. Intraperitoneal delivery providedsimilar plasma and intraocular concentrations of the drugs.

Alternatively, daily oral dosages of TEMPOL-H could be provided inamounts of 100, 1000 or 6000 mg per 100 kg body weight. The dosageswould preferably be given in divided doses, for example four time perday. The oral dosages could be added to food or water. Blood or anteriorchamber concentrations of TEMPOL-H can be determined by EPR to adjustthe dosage to a desired intraocular concentration.

For the purpose of oral or parenteral administration, the activeingredient may be incorporated into tablets, pills, capsules and thelike, which may also contain one or more of the following adjuvants:binders, such as microcrystalline cellulose, gum tragacanth or gelatin;excipients, such as starch or lactose, disintegrating agents such asalginic acid, corn starch and the like; lubricants, such as magnesiumstearate; glidants, such as colloidal silicon dioxide; and sweeteningagents, such as sucrose. When the dosage unit form is a capsule, it maycontain, in addition to the materials of the above type, a liquidcarrier such as polyethylene glycol or a fatty oil.

EXAMPLE 8

Systemic Delivery of Reducing Agent

The reducing agent MPG was also delivered to the subjects bysubcutaneous, intraperitoneal and ocular routes, as described in Example7, through an implanted osmotic pump. A 2 ml volume of 100 mM MPG wasprovided in the pump and delivered over a period of 7 days or 14 days.The MPG was detected in the eye after administration by these routes.

EXAMPLE 9

Concomitant Delivery of Hydroxylamine and Reducing Agent

In those embodiments wherein combined delivery of the hydroxylamine andreducing agent is desired, the hydroxylamine and reducing agent can beadministered either separately or in combination. The combined drugscould be delivered, for example, by direct intraocular injection orsubconjunctival deposition, or oral ingestion. Alternatively, thehydroxylamine and reducing agent can be delivered separately but atabout the same time (for example within about 1 minute of each other).

The delivery of the hydroxylamine could be achieved by any of the routesdescribed in Example 7, and the reducing agent could also be deliveredby any of the routes described in Example 8. Hence the TEMPOL-H could bedelivered intraperitoneally as described in Example 7, while the MPGcould be delivered subcutaneously as in Example 8.

Another approach to delivering the drugs is to provide the hydroxylaminein the form of a nitroxide radical prodrug that is subsequently reducedto the hydroxylamine after moving through the cornea into the anteriorchamber. In this example, 100 mM concentration TEMPOL would beadministered to the eye in a 50 μL drop instilled in the inferior fornixof the eye. After a period of 5-10 minutes (during which the TEMPOL isallowed to pass through the cornea), 100 mM concentration MPG would bedelivered to the eye in a 50 μL drop that is also instilled in theinferior fornix of the eye. The MPG would then pass separately throughthe cornea, where it would reduce TEMPOL to TEMPOL-H in the aqueoushumor of the anterior chamber of the eye.

Some reducing agents, such as N-acetyl-cysteine, do not quickly passthrough the cornea. In this situation, the reducing agent may also beadministered as a prodrug that is rapidly transported across the cornealbarrier into the aqueous humor of the anterior chamber, as in U.S. Pat.No. 5,519,054. Alternatively, the N-acetyl-cysteine could be givenorally.

The delivery of drug to the anterior chamber would be repeated daily forthe duration of treatment, which could extend throughout the life of thepatient, or until surgical extraction and replacement of the lens isrequired.

The invention being thus described, variation in the materials andmethods for practicing the invention will be apparent to one of ordinaryskill in the art. Such variations are to be considered within the scopeof the invention, which is set forth in the claims below.

REFERENCES

Various articles of the scientific and patent literature are citedthroughout the instant document. Each cited article is herebyincorporated by reference in its entirety.

1. B. Halliwell and J. M. C. Gutteridge, Free Radicals in Biology andMedicine, 2nd Ed., c. 1989 by Clarendon Press, Oxford.

2. Oxygen Radicals: Systemic Events and Disease Processes, D. K. Das andW. B. Essman, eds., c. 1990 by Karger, Basel, Switzerland.

3. C. A. Rice-Evans and A. T. Diplock, Free Radical Biology & Medicine,15:77 (1993).

4. H. Nishigari, Investigative Ophthalmology & Visual Science, 25:1051(1984).

5. U. A. Nilsson et al., J. Biol. Chem., 19:11131 (1989).

6. A. Samuni et al., pp. 85-92 in Antioxidants in Therapy and PreventiveMedicine, I. Emerit et al., eds. c. 1990 by Plenum Press, New York, N.Y.

7. A. Samuni et al., J. Clin. Invest., 87:1526 (1991).

8. A. Samuni et al., Biochemistry, 30:555 (1991).

9. J. R. Reddan et al, Exp. Eye Res., 56:543 (1993).

10. S. J. Tumminia et al., Exp. Eye Res., 58:367 (1994).

11. J. S. Zigler et el., Investigative Ophthalmology & Visual Science30:2195 (1989).

12. Mitchell et al., Arch. Biochem. Biophys., 289:62-70 (1991).

13. Krishna et al., Cancer Research, 51:6622-6628 (1991).

We claim:
 1. A pharmaceutical composition comprising a hydoxylaminecompound in a therapeutically sufficient amount to prevent or retard thedevelopment of a cataract in a subject to whom the composition isadministered, the composition further comprising a reducing agent formaintaining the hydroxylamine in a reduced form.
 2. The composition ofclaim 1, wherein the reducing agent is a sulfhydryl compound that ispresent in a sufficient amount to maintain substantially all thehydroxylamine compound in a reduced state.
 3. The composition of claim2, wherein the reducing agent is selected from the group consisting ofmercaptopropionyl glycine, N-acetyl cysteine, β-mercaptoethylamine andglutathione.
 4. The composition of claim 1, wherein the hydroxylaminecompound is selected from the group consisting of TEMPOL-H, TEMPO-H andOXANO-H.
 5. The composition of claim 3, wherein the sulfhydryl compoundis mercaptopropionyl glycine.
 6. The composition of claim 5 wherein thehydroxylamine compound is TEMPOL-H.
 7. The composition of claim 1,wherein the composition is a topical preparation suitable forinstillation in the human eye.
 8. The composition of claim 7, whereinthe composition is in a dosage unit form suitable for instillation in oron the human eye.
 9. The composition of claim 6, wherein the compositioncomprises TEMPOL-H in an amount that is sufficient to provide TEMPOL-Hin a blood concentration of about 25 μM to 3 mM, and mercaptopropionylglycine in an amount sufficient to provide mercaptopropionyl glycine ina blood concentration of about 0.25 to 50 mM.
 10. The composition ofclaim 6, wherein the composition comprises TEMPOL-H in an amount that issufficient to provide TEMPOL-H in a concentration of about 1 μM to 1 mMin aqueous humor of the eye, and mercaptopropionyl glycine in an amountsufficient to provide mercaptopropionyl glycine in a concentration ofabout 0.1 to 5 mM in aqueous humor of the eye.
 11. A pharmaceuticalcomposition comprising a hydroxylamine compound and a reducing agent,for instillation in a human eye to retard or prevent the development ofa cataract in a crystalline lens of the eye.
 12. The composition ofclaim 9, where the hydroxylamine compound comprises TEMPOL-H in anamount that is sufficient to provide TEMPOL-H in a concentration ofabout 1 μM to 1 mM in aqueous humor of the eye, and mercaptopropionylglycine in an amount sufficient to provide mercaptopropionyl glycine ina concentration of about 0.1 to 5 mM in aqueous humor of the eye.
 13. Amethod for inhibiting or preventing the development of a cataract orinhibiting the progression of an incipient cataract which comprisesadministering to a patient a hydroxylamine compound and a reducing agentin a therapeutically sufficient amount to prevent or retard thedevelopment of a cataract in a subject to whom the composition isadministered.
 14. The method of claim 13, wherein the step ofadministering the composition comprises administering TEMPOL-H as thehydroxylamine compound.
 15. The method of claim 14, wherein the step ofadministering the composition comprises administering mercaptopropionylglycine as the reducing agent.
 16. The method of claim 13, wherein thestep of administering the composition comprises administering TEMPOL-Hin an amount that is sufficient to provide TEMPOL-H in a concentrationof about 1 μM to 1 mM in aqueous humor of the eye, and mercaptopropionylglycine in an amount sufficient to provide mercaptopropionyl glycine ina concentration of about 0.1 to 5 mM in aqueous humor of the eye. 17.The method of claim 13, wherein the step of administering thecomposition comprises administering the composition by adsorption into amaterial selected from the group consisting of a soft contact lens and acollagen corneal shield, and applying said material to the eye.
 18. Themethod of claim 15, wherein the step of administering the compositioncomprises administering the composition directly to the eye in a topicalform selected from the group consisting of eye drops and ointment.
 19. Apharmaceutical composition for retarding the development of cataracts ina subject, comprising TEMPOL-H in a therapeutically sufficient amount toprevent or retard the development of a cataract in a subject to whom thecomposition is administered, a reducing agent comprisingmercaptopropionyl glycine in a sufficient amount to maintain theTEMPOL-H substantially completely reduced, and a pharmaceutical carriersuitable for instillation in the human eye.